Enhanced human antigen-specific B cell responses using in vitro 3D tonsil cultures containing stromal cells

The structural complexity of secondary lymphoid organs (SLOs) and their role in shaping antigen-specific B cell responses, pose significant challenges in modeling human germinal center (GC) response in vitro. A human 3D lymphoid model incorporating lymphoid and stromal cell types recapitulates key immune and structural features, enabling the study of antigen-specific B and T cell interactions beyond current 2D culture limitations. In this study, human tonsil cells were cultured with and without tonsil-derived fibroblastic reticular cells (FRCs) either in 2D or within a 3D PEG-4MAL hydrogel culture. Antigen-specific B cell responses in co-cultures were studied by comparing unstimulated cultures to stimulation with antigen (SARS-CoV-2 spike (S) or Influenza hemagglutinin (HA), both with or without adjuvant R848), S-nanoparticles and influenza vaccines. Combination of FRCs with the 3D matrix significantly improved B and T cell survival and facilitated reaggregation into follicle-like structures. Antigen-specific responses were most pronounced in 3D FRC-supported co-cultures, with increasing S- or HA-specific B cell frequencies, antibody-secreting cell differentiation, and secretion of antigen-specific antibodies. Importantly, cell death and unspecific bystander activation was lowest in 3D FRC-supported cultures. Additionally, GC-associated chemokine receptors CXCR4 and CXCR5 showed distinct expression patterns on CD27+ CD38+ B cells, reflecting GC-like dark and light zone organization typically observed in SLOs in vivo. Autologous and allogeneic FRC-supported cultures yielded comparable results, demonstrating the platforms potential for high-throughput applications. The 3D FRC-supported lymphoid cultures offer a physiologically relevant platform for studying human GC responses in vitro, supporting mechanistic research into adaptive immunity and enabling the screening of vaccine immunogens and adjuvants in a controlled setting.